PDRN, preparation method and application of PDRN in ulcerative colitis

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to PDRN, a preparation method and application of the PDRN in ulcerative colitis. The PDRN preparation method comprises the following steps: adding salmon sperms into a solvent, crushing to obtain a solid-liquid mixture, centrifuging, collecting precipitate, adding a mixed salt solution, and reacting to obtain a reaction solution; adding a compound enzyme into the reaction liquid, and performing enzymolysis to obtain enzymatic hydrolysate; and removing residual impurities after enzymolysis, carrying out alcohol precipitation, collecting the precipitate, washing the precipitate, centrifuging, collecting the precipitate, crushing the precipitate, and freeze-drying to obtain the PDRN. The PDRN provided by the invention has no cytotoxicity, and can significantly inhibit JAK / STAT and NF [kappa] B inflammation signal pathways and reduce the level of proinflammatory factors; the traditional Chinese medicine composition can improve weight loss, colon shortening, mucosal lesion and other symptoms caused by ulcerative colitis, reduce histological score and inflammatory cell infiltration, and play a synergistic treatment role by adjusting the diversity of intestinal flora.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a PDRN, its preparation method, and its application in ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease of unknown etiology, primarily affecting the rectal and colonic mucosa. Clinical manifestations include persistent or recurrent diarrhea, bloody and mucous stools, abdominal pain, and tenesmus. Severe cases can be complicated by intestinal perforation, toxic megacolon, and even cancer, posing a significant threat to patients' quality of life and health. Currently, the pathogenesis of UC is not fully understood, but it is generally believed to be closely related to the synergistic effects of genetic susceptibility, immune imbalance, gut microbiota dysbiosis, and environmental factors (such as diet and infection). Abnormal immune activation (such as overactivation of inflammatory signaling pathways like JAK / STAT and NFκB) and damage to the intestinal mucosal barrier are considered the core pathological links.

[0003] Currently, the clinical treatment of UC primarily aims to control inflammation, alleviate symptoms, and prevent relapse. Commonly used drugs include aminosalicylic acid derivatives, glucocorticoids, immunosuppressants (such as azathioprine), and biologics (such as anti-TNF-α monoclonal antibodies). However, existing treatment options have significant limitations: aminosalicylic acid derivatives are only effective for mild to moderate cases, and long-term use easily leads to drug resistance; glucocorticoids have significant short-term efficacy, but long-term use can cause serious side effects such as osteoporosis and increased risk of infection; while immunosuppressants and biologics can improve the prognosis of severely ill patients, they suffer from slow onset of action, high cost, large individual variability in response, and potential immunosuppression-related complications (such as opportunistic infections and increased risk of lymphoma). Therefore, the search for novel anti-UC drugs that are safe, have clear efficacy, and have few side effects has become a research hotspot in the current biomedical field.

[0004] Natural products, due to their diverse structures and broad bioactivities, exhibit unique advantages in the treatment of inflammatory diseases. Among them, polydeoxyribonucleotides (PDRN), a bioactive substance extracted from sources such as salmon sperm, has been proven to have anti-inflammatory, tissue repair-promoting, and immune-regulating effects. Studies have shown that PDRN can exert protective effects in various inflammatory models by inhibiting the release of pro-inflammatory factors (such as IL-6 and TNF-α), regulating macrophage polarization, and improving intestinal mucosal barrier function. However, current PDRN extraction processes often suffer from insufficient purity and unstable activity, and its specific mechanisms of action against ulcerative colitis (UC) (such as its regulatory effects on gut microbiota and its targeting of key inflammatory signaling pathways) are not yet fully understood, limiting its clinical application value.

[0005] Patent CN116891847B provides a method for preparing zinc deoxyribonucleic acid (PDRN) salt with skin barrier repair effects. The method includes: taking fish testes and washing them with SSC buffer; adding the obtained fish spores to SSC buffer, homogenizing them into a homogenate, adding more SSC buffer, and centrifuging until the supernatant is clear; digesting the homogenate in a water bath, adding ethanol to the supernatant to obtain sodium PDRN; adding the sodium PDRN solution to the zinc salt solution, adjusting the pH, stirring, allowing it to stand, centrifuging, and drying to obtain zinc PDRN salt. This method reduces the number of steps in PDRN preparation while controlling the temperature during extraction to avoid localized high temperatures that could affect PDRN quality. Simultaneously, the method directly converts the zinc salt into PDRN zinc salt precipitate through ion exchange to adjust the pH in the zinc salt solution.

[0006] Patent CN120082544A discloses a method for preparing PDRN: Step 1, collecting materials and pyrolyzing them to prepare a lysate; Step 2, adding salt and a protein denaturing agent to the lysate for salting out to prepare a clear salt-out solution; Step 3, adding an organic solvent to the clear salt-out solution to precipitate and prepare a crude product; Step 4, drying the crude product to obtain the final product. Compared to the prior art of extracting PDRN from salmon semen, the PDRN preparation method disclosed in this invention is simpler and more convenient.

[0007] However, in existing PDRN preparation processes, the selection of the enzymatic hydrolysis system, the efficiency of impurity removal, and the optimization of the purification process remain key issues that urgently need to be addressed. Therefore, developing a PDRN derivative with optimized process, high purity, strong activity, and the ability to exert anti-UC effects through multiple targets (such as inflammatory pathways and gut microbiota) has significant theoretical implications and promising clinical application prospects. Summary of the Invention

[0008] To address the above shortcomings, this invention provides a PDRN, its preparation method, and its application in ulcerative colitis.

[0009] the term: 1. PDRN: Polydeoxyribonucleotide, is a nucleic acid substance formed by the polymerization of deoxyribonucleotides. It is mostly extracted from sources such as salmon sperm and has certain anti-inflammatory and tissue repair-promoting biological activities.

[0010] This invention specifically refers to a polydeoxyribonucleotide (PDRN) extracted from salmon sperm, prepared through a specific process (including solvent disruption, mixed salt solution treatment, complex enzymatic hydrolysis, impurity removal, alcohol precipitation purification, and freeze-drying). It possesses anti-inflammatory activity, inhibiting inflammatory signaling pathways such as JAK / STAT and NFκB, regulating gut microbiota, and playing a role in the treatment of ulcerative colitis. The PDRN-1 of this invention was purchased from PharmaResearch Co., Ltd. as a positive control for experimental comparison.

[0011] 2. PN: Polynucleotide, a biological macromolecule composed of multiple nucleotides linked by phosphodiester bonds. In this invention, it serves as a positive control to compare its activity with that of PDRN. The difference between PN and PDRN is that PN has a molecular weight exceeding 1500 kDa, while PDRN is a polymer between 5 and 1500 kDa.

[0012] 3. Solid-liquid ratio: This refers to the ratio between the mass of a solid substance (usually expressed in "g") and the volume of a liquid substance (usually expressed in "mL"). In this invention, it is used to describe the ratio of solid to liquid in systems such as salmon sperm and solvent, precipitate and mixed salt solution. For example, a "1:6 solid-liquid ratio" means that 1g of solid corresponds to 6mL of liquid.

[0013] 4. Crushing: In this invention, this specifically refers to the process of breaking down the cellular structure of salmon sperm through physical methods (such as processing with a high-speed blender), making it easier for the target components (such as nucleic acids) to be released into the solvent, forming a solid-liquid mixture. The purpose is to improve the efficiency of subsequent extraction. This invention does not limit the method of crushing.

[0014] 5. Freeze-drying: Freeze-drying is a process in which wet materials or solutions are frozen into a solid state at low temperatures, and then the water in the solid is directly sublimated into a gaseous state under vacuum to obtain a dried product. In this invention, it is used to dry purified PDRN precipitate to maintain its biological activity and facilitate storage and subsequent use.

[0015] 6. Enzymatic hydrolysis: This refers to the process of breaking down biological macromolecules (such as proteins and nucleic acids) into smaller fragments using the catalytic action of enzymes under specific conditions (such as temperature and pH). In this invention, a complex enzyme composed of trypsin and papain is used to enzymatically hydrolyze relevant components in salmon sperm to release and prepare the target product PDRN.

[0016] The technical solution of this invention is as follows: On one hand, the present invention provides a PDRN, the preparation method of which includes the following steps: S1. Salmon sperm is added to a solvent and broken down to obtain a solid-liquid mixture; S2. Centrifuge the solid-liquid mixture, collect the precipitate, add the mixed salt solution, react, and obtain the reaction solution; S3. Add the compound enzyme to the reaction solution and perform enzymatic hydrolysis to obtain the enzymatic hydrolysate; S4. The enzyme hydrolysate removes the impurities remaining after enzymatic hydrolysis. After alcohol precipitation, the precipitate is collected, washed, centrifuged, and the precipitate is collected. The precipitate is then broken up and freeze-dried to obtain PDRN.

[0017] Specifically, the solvent mentioned in step S1 includes any one or more of the following: physiological saline, water, phosphate buffer, citrate buffer, acetate buffer, HEPES buffer, Tris-HCl buffer, and Tris buffer.

[0018] Preferably, the solvent in step S1 is physiological saline.

[0019] Specifically, the amount of solvent added in step S1 is 5-10 mL per 1 g of salmon sperm.

[0020] Preferably, the amount of solvent added in step S1 is 5-6 mL, 6-7 mL, 7-8 mL, 8-9 mL or 9-10 mL per 1 g of salmon sperm.

[0021] More preferably, the amount of solvent added in step S1 is: 6 mL of solvent per 1 g of salmon sperm.

[0022] Specifically, the crushing described in step S1 includes, but is not limited to: crushing with a wall-breaking machine and / or ultrasonic crushing.

[0023] Preferably, the crushing in step S1 is performed by a wall-breaking machine.

[0024] Specifically, the centrifugation in step S2 includes centrifugation at 0-4℃ and 2000-5000rpm for 20-40 minutes.

[0025] Preferably, the centrifugation in step S2 includes centrifugation at 4°C and 3500 rpm for 30 min.

[0026] Specifically, the mixed salt solution mentioned in step S2 is a mixed solution of NaCl and EDTA, wherein the molar ratio of NaCl to EDTA in the mixed salt solution is 0.1-0.5:1.

[0027] Preferably, the molar ratio of NaCl to EDTA in the mixed salt solution in step S2 is 0.1-0.2:1, 0.2-0.3:1, 0.3-0.4:1, or 1, 0.4-0.5:1.

[0028] More preferably, the molar ratio of NaCl to EDTA in the mixed salt solution described in step S2 is 0.3:1.

[0029] Specifically, the amount of mixed salt solution added in step S2 is: 1g of the precipitate in step S2 is added to 3-7mL of mixed salt solution.

[0030] Preferably, the amount of mixed salt solution added in step S2 is: 1g of the precipitate in step S2 is added to 3-5mL, 5-6mL or 6-7mL of mixed salt solution.

[0031] More preferably, the amount of mixed salt solution added in step S2 is: 1g of the precipitate in step S2 is added to 5mL of mixed salt solution.

[0032] Specifically, the reaction time in step S2 is 5-15 minutes.

[0033] Preferably, the reaction time in step S2 is 5-6 min, 6-7 min, 7-8 min, 8-9 min, 9-10 min, 10-11 min, 11-12 min, 12-13 min, 13-14 min, or 14-15 min.

[0034] More preferably, the reaction time in step S2 is 10 minutes.

[0035] Specifically, the complex enzyme mentioned in step S3 is a mixture of trypsin and papain, wherein the mass ratio of trypsin to papain is 2-3:1.

[0036] Preferably, the complex enzyme in step S3 is a mixture of trypsin and papain, wherein the mass ratio of trypsin to papain is 2.0-2.1:1, 2.1-2.2:2.2-2.3:1, 2.3-2.4:1, 2.4-2.5:1, 2.5-2.6:1, 2.6-2.7:1, 2.7-2.8:1, 2.8-2.9:1, or 2.9-3.0:1.

[0037] More preferably, the complex enzyme in step S3 is a mixture of trypsin and papain, wherein the mass ratio of trypsin to papain is 2.5:1.

[0038] Specifically, the mass ratio of the complex enzyme mentioned in step S3 to the salmon sperm mentioned in step S1 is 1:200-300.

[0039] Preferably, the mass ratio of the complex enzyme in step S3 to the salmon sperm in step S1 is 1:200-210, 1:210-220, 1:220-230, 1:230-240, 1:240-250, 1:250-260, 1:260-270, 1:270-280, 1:280-290, or 1:290-300.

[0040] Specifically, the enzymatic hydrolysis reaction conditions described in step S3 are 35-40℃ for 15-25 hours.

[0041] Preferably, the enzymatic hydrolysis reaction conditions in step S3 are 35-36℃, 36-37℃, 37-38℃, 38-39℃ or 39-40℃ for 15-16h, 16-17h, 17-18h, 18-19h, 19-20h, 20-21h, 21-22h, 22-23h, 23-24h or 24-25h.

[0042] More preferably, the enzymatic hydrolysis reaction conditions in step S3 are 37°C for 20 hours.

[0043] Specifically, the step of removing residual impurities after enzymatic hydrolysis in step S4 includes: adding SDS and NaCl to the enzymatic hydrolysate, and collecting the supernatant by centrifugation after the reaction; the alcohol precipitation step in step S4 includes: adding ethanol to the supernatant.

[0044] Preferably, the amount of SDS added is 10%-15% of the mass of salmon sperm.

[0045] More preferably, the amount of SDS added is 10%-11%, 11%-12%, 12%-13%, 13%-14% or 14%-15% of the salmon sperm mass.

[0046] More preferably, the amount of SDS added is 10% of the mass of salmon sperm.

[0047] Preferably, the final concentration of NaCl is 1.5-2.0M.

[0048] More preferably, the final concentration of NaCl is 1.5-1.6M, 1.6-1.7M, 1.7-1.8M, 1.8-1.9M or 1.9-2.0M.

[0049] More preferably, the final concentration of NaCl is 1.6M.

[0050] Preferably, the reaction is a stirred reaction for 3-5 hours.

[0051] More preferably, the reaction is a stirred reaction for 4 hours.

[0052] Preferably, the centrifugation includes centrifugation at 5000-7000 rpm for 30-90 minutes.

[0053] More preferably, the centrifugation includes centrifugation at 6000 rpm for 60 minutes.

[0054] Preferably, the ethanol is 90%-100% v / v ethanol; the volume ratio of ethanol to supernatant is 1-3:1.

[0055] More preferably, the ethanol is 100% v / v ethanol.

[0056] More preferably, the volume ratio of ethanol to supernatant is 1-2:1 or 2-3:1.

[0057] More preferably, the volume ratio of ethanol to supernatant is 2:1.

[0058] Specifically, the washing described in step S4 involves washing with ethanol 2-3 times.

[0059] Preferably, the washing in step S4 involves washing twice with 70%-80% v / v ethanol.

[0060] More preferably, the washing in step S4 involves washing twice with 75% v / v ethanol.

[0061] Specifically, the centrifugation described in step S4 is centrifugation at 2000-6000 rpm for 5-10 minutes.

[0062] Preferably, the centrifugation in step S4 is centrifugation at 4000 rpm for 5 minutes.

[0063] Specifically, the crushing described in step S4 includes, but is not limited to: crushing with a wall-breaking machine and / or ultrasonic crushing.

[0064] Preferably, the crushing in step S4 is crushing using a wall-breaking machine.

[0065] On the other hand, the present invention provides a method for preparing PDRN as described in any of the above claims.

[0066] In another aspect, the present invention provides the application of PDRN as described in any of the above claims in the preparation of products, wherein the products are drugs for the prevention, treatment or adjunctive treatment of colitis.

[0067] Specifically, the colitis mentioned includes any one or more of infectious colitis, ischemic colitis, pseudomembranous colitis, ulcerative colitis, and Crohn's disease of the colon.

[0068] Preferably, the colitis is ulcerative colitis.

[0069] In another aspect, the present invention provides a medicine comprising any of the PDRNs described above.

[0070] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0071] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, one or more combinations of wetting agents, emulsifiers, preservatives, antioxidants, buffers, diluents, lubricants, solutes, suspending agents, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0072] Specifically, the dosage form of the drug includes gastrointestinal dosage forms or non-gastrointestinal dosage forms.

[0073] Preferably, the gastrointestinal dosage forms include, but are not limited to: tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0074] Preferably, the non-gastrointestinal dosage forms include, but are not limited to: injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0075] In another aspect, the present invention provides a method for treating colitis, the method comprising using any of the PDRNs or drugs described above.

[0076] Specifically, the method includes administering an effective amount of PDRN or a drug to the subject.

[0077] Preferably, the subject is a mammal.

[0078] More preferably, the subject is a human being.

[0079] Specifically, the colitis mentioned includes any one or more of infectious colitis, ischemic colitis, pseudomembranous colitis, ulcerative colitis, and Crohn's disease of the colon.

[0080] Preferably, the colitis is ulcerative colitis.

[0081] The beneficial effects of this invention are as follows: 1. This invention achieves efficient extraction of PDRN through steps such as pretreatment with a mixed salt solution in a specific ratio, compound enzymatic hydrolysis, removal of impurities, and purification by ethanol precipitation. The process conditions are mild, which can maximize the preservation of the product's biological activity, and the product has high purity (verified by HPLC and infrared spectroscopy as a polydeoxyribonucleotide).

[0082] 2. Cytotoxicity experiments showed that the PDRN of the present invention was non-toxic to BJ cells, HUVEC cells, and L929 cells at a concentration of 100-1000 μg / mL, and could promote cell proliferation, demonstrating its good biocompatibility and laying a safe foundation for its subsequent application as a drug.

[0083] 3. The PDRN of the present invention can significantly inhibit the JAK / STAT (downregulate STAT2, STAT3 phosphorylation and JAK2, JAK3 activity) and NFκB (reduce IKK phosphorylation) signaling pathways, reduce the expression of LPS-induced pro-inflammatory factors such as IL-6, TNF-α, IL-1β, and CXCL10, and block the inflammatory cascade response from the source.

[0084] 4. In a DSS-induced ulcerative colitis mouse model, the PDRN of this invention can significantly reduce the disease activity index (DAI), alleviate weight loss, increase colon length, reduce mucosal erosion, crypt shortening and inflammatory cell infiltration, and reduce histological scores. The effect is better than the positive control drugs PN and PDRN-1.

[0085] 5. The PDRN of this invention reduces the expression of inflammatory proteins such as IL-1β and CD11b in colonic tissue, reduces the proportion of CD45+ immune cells and activated myeloid cells (MCHII / 7AAD+) in the intestinal mucosa, and restores intestinal immune homeostasis.

[0086] 6. The PDRN of this invention can reverse DSS-induced intestinal flora imbalance: increase flora α diversity (ACE, Chao1 index), reduce flora imbalance index (MDI), increase the abundance of beneficial bacteria (such as succinic acid-producing Parvibacter, anti-inflammatory Adlercreutzia, and short-chain fatty acid-producing Lachnospiraceae_NK4A136_group), and enhance the therapeutic effect on ulcerative colitis through the synergistic effect of the "flora-immunity-metabolism" axis. Attached Figure Description

[0087] Figure 1 The figure shows the preparation and determination of PDRN; A in the figure is the high performance liquid chromatogram of PDRN; B is the infrared spectrum of PDRN.

[0088] Figure 2The figure shows the cytotoxicity of PDRN and its inhibitory effects on the JAK / STAT and NFκB signaling pathways; A represents the cytotoxicity of PDRN on BJ cells; B represents the cytotoxicity of PDRN on L929 cells; C represents the cytotoxicity of PDRN on HUVEC cells; D represents the relative mRNA level of IL-6 in peritoneal macrophages pretreated with PDRN solution and then stimulated with LPS; E represents the relative mRNA level of peritoneal macrophages after pretreatment with PDRN solution and then stimulated with LPS; F represents the relative mRNA level of CXCL10 in peritoneal macrophages after pretreatment with PDRN solution and then stimulated with LPS; G represents... The relative mRNA level of IL-1β in peritoneal macrophages after pretreatment with PDRN solution followed by LPS stimulation; H represents the IKK level and phosphorylation of peritoneal macrophages after pretreatment with PDRN solution followed by LPS stimulation, detected by Western blotting; I represents the STAT2 level and phosphorylation of peritoneal macrophages after pretreatment with PDRN solution followed by IFN-β stimulation, detected by Western blotting; J represents the STAT3, JAK2, and JAK3 levels and phosphorylation of peritoneal macrophages after pretreatment with PDRN solution followed by IL-6 stimulation, detected by Western blotting; * P < 0.05, ** P < 0.01, *** P < 0.001 in the figure.

[0089] Figure 3 To assess the effect of PDRN on improving clinical symptoms in UC model mice; A in the figure is a macroscopic image of the colon; B is the quantitative result of colon length; C is the DAI score; D is the percentage of weight recovery in mice; F is the histological score; E is the percentage of involvement; G is a representative image of colon H&E staining; * P<0.05, *** P<0.001, **** P<0.0001 in the figure.

[0090] Figure 4 The effect of PDRN on the expression of inflammation-related proteins in UC model mice is shown in the figure. Figure A shows the immunofluorescence assay of IL-1β and Cd11b expression in colon tissue. Colon tissue sections were immunostained to reveal different molecules (green IL-1β, red Cd11b), and a representative micrograph was captured using an inverted fluorescence microscope (scale bar = 50 μm). Figure B shows the positive area analysis of Cd11b immunofluorescence staining results. Figure C shows the positive area analysis of IL-1β immunofluorescence staining results. Figure D shows the percentage of CD45+ cells analyzed by flow cytometry. Figure E shows the percentage of MCHII / 7AAD+ cells analyzed by flow cytometry. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0091] Figure 5The role of PDRN in the inflammatory response in the transcriptome analysis of the UC model is shown in the figure. A represents the top 20 KEGG classification terms; B represents the relative mRNA level of CXCL-2 measured by RT-PCR; C represents the relative mRNA level of IL-1β measured by RT-PCR; D represents the relative mRNA level of TNF-α measured by RT-PCR; E represents the relative mRNA level of CCL-2 measured by RT-PCR. In the figure, ** P < 0.01, **** P < 0.0001.

[0092] Figure 6 The figure shows the effect of PDRN on the gut microbiota of UC model mice; A in the figure represents the β diversity of gut microbiota at the species level as assessed by the PCoA method; B represents the change in the number of ASVs in the normal control UC model group and the PDRN treatment group as the sample size increases; C represents the gut microbiota dysbiosis index (MDI); D represents α diversity represented by the ACE index; and E represents α diversity represented by the Chao index.

[0093] Figure 7 The effect of PDRN on the gut microbiota of UC model mice was investigated; community structure bar chart analysis was used to display the composition of the microbial community, and the relative abundance of various microorganisms in different samples was displayed intuitively through the bar chart.

[0094] Figure 8 The effect of PDRN on IL-6 expression levels in comparative examples 1-6. Detailed Implementation

[0095] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0096] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0097] Materials and reagents: Cell lysis buffer (catalog number 9803) was purchased from Cell Signaling Technology. Protease inhibitors (catalog number 11836145001), a mixture of phosphorylation inhibitors (catalog number 4906837001), and SYBR Green PCR MasterMix (2×) (catalog number 4913914001) were purchased from Roche Diagnostics. Horseradish peroxidase-labeled secondary antibodies (catalog numbers abs20001 and abs20002) were purchased from Absin Biotech. Kolliphor® HS 15 (HS-15) (catalog number 42966) and lipopolysaccharide / LPS (catalog number 916374) were purchased from Sigma-Aldrich. Recombinant mouse IL-6 (catalog number 200-06) and IFN-β (catalog number 300-02BC) were purchased from PeproTech.

[0098] Statistical analysis: All results in this invention are expressed as mean ± standard error. Independent unpaired two-tailed t-tests were used to compare two groups, and univariate ANOVA with Dunnett's test was used to compare multiple groups. P < 0.05 was considered statistically significant. Results are expressed as mean ± standard deviation (SD). All statistical analyses were performed using GraphPad (Prism 9.00). Two-tailed Student's t-tests were used to assess statistical significance between two groups. P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001). All data presented in this study are from at least three independent experiments.

[0099] Example 1: Preparation of PDRN 1. Preparation of PDRN: PDRN was prepared using an enzymatic hydrolysis method. The specific steps for PDRN extraction are as follows: S1. After washing the thawed salmon sperm with physiological saline, use a high-speed blender to add physiological saline at a solid-liquid ratio of 1:6 to fully break down the salmon sperm and obtain a solid-liquid mixture. S2. Centrifuge the solid-liquid mixture at 4℃ and 3500rpm for 30min, discard the supernatant, add 100g of precipitate to 500mL of mixed salt solution (0.15M NaCl / 0.5 mM EDTA) at 37℃, and react with stirring for 10min. S3. Add the compound enzyme at a ratio of 250:1 (mass ratio of salmon sperm to compound enzyme (trypsin:papain in a mass ratio of 2.5:1) and hydrolyze at 37°C for 20 hours with stirring to obtain the enzymatic hydrolysate. S4. After enzymatic hydrolysis, add 10% SDS by weight of salmon sperm and NaCl to the hydrolysate, and continue stirring for 4 h; centrifuge at 6000 rpm for 1 h at room temperature and collect the supernatant.

[0100] S5. Add pre-cooled anhydrous ethanol to the supernatant at a volume ratio of 2:1, stir, and precipitate flocculated PDRN at 4°C. Wash twice with 75% ethanol, centrifuge at 4000 rpm for 5 min, discard the supernatant, remove the residual ethanol with a pipette tip, crush the precipitate, and freeze-dry it in a freeze dryer to obtain the PDRN of this invention. After freeze-drying, dissolve it in physiological saline to obtain the PDRN solution.

[0101] 2. Determination of the relative molecular mass of PDRN The relative molecular mass of PDRN was determined by high-performance gel permeation chromatography (HPGPC). PDRN was prepared at a concentration of 5 mg / mL, and its relative molecular mass was measured on a Tskgel GM PWxl gel column with an injection volume of 20 μL. Dextran was used as a control, and the chromatographic retention times of each standard component were determined. A standard curve was plotted against the logarithm of the standard retention time (t) and its molecular weight (Log Mp), yielding a linear regression equation for molecular weight versus retention time. The linear regression equation was: Standard curve Log(Mw) = -0.5216tR + 13.359 (R² = 0.9952). The HPGPC elution curve of the test sample PDRN is shown below. Figure 1 As shown in A in the figure. The sample contained the main quantum dot macromolecular compound component at 15.017 min, and the relative molecular mass of the main component was calculated to be approximately 335 kDa based on the plotted standard curve.

[0102] 3. Structural confirmation of PDRN compounds PDRN is an abbreviation for polydeoxyribonucleotide. Literature reports it as a class of small molecule compounds composed of purine or pyrimidine bases, deoxyribose, and phosphate. Specifically, it comprises four bases: adenine (A), thymine (T), cytosine (C), and guanine (G). This invention utilizes infrared spectroscopy to confirm the structure of PDRN compounds.

[0103] Using infrared methods ( Figure 1 In section B), the structure of the PDRN macromolecular compound component in the product was confirmed, and it was found that PDRN possesses the properties of a ribonucleotide. As shown in the figure, 3347 cm⁻¹ -1 3200cm -1 The peaks are the stretching vibration peaks of OH and NH, respectively; 1525 and 1485, 1115 and 1371 cm⁻¹.-1 Vibrational absorption peaks for CN and NH, respectively; 1232 cm⁻¹ -1 This is the P=O vibration peak of nucleic acids; 1080 cm⁻¹ -1 For -CO vibration, -POC vibration, -PO, and -C P=O vibration; 1080cm -1 These are the vibrational peaks of -CO, -POC, -PO, and -CO (DNA, phospholipids).

[0104] Example 2: PDRN is non-cytotoxic and has a good inhibitory effect on both the JAK / STAT and NFκB inflammatory signaling pathways. 1. Determination of the cytotoxicity of resazurin (sodium) to PDRN The cytotoxicity of the drug to BJ cells, HUVEC cells, and L929 cells was detected using the resazurin assay (Sigma, USA). After the cells were fully attached, PDRN, or PN (purchased from PharmaResearch Co., Ltd., catalog number DJU, denoted as DJ), or PDRN-1 (purchased from PharmaResearch Co., Ltd., catalog number DCELL-I, denoted as DC) from Example 1 was added to 96-well plates at final concentrations of 100 μg / mL, 500 μg / mL, and 1000 μg / mL, with cell culture medium used as a blank control. Meanwhile, 100 μL of PBS was added to the surrounding wells and incubated for 48 h. Then, 10 μL of leaf azurite (1 mg / mL) was added to each well and incubated in the dark for 3-4 h. The absorbance values ​​were read at 0 h, 24 h and 48 h using a multi-functional microplate reader (SpectraMax I3) under the conditions of excitation at 549 nm and emission at 595 nm. The data were recorded, statistically analyzed and plotted.

[0105] like Figure 2 As shown in AC, PDRN at concentrations of 100, 500, and 1000 μg / mL showed no significant toxicity to human BJ cells, HUVECs, and L929 cells. At the same time, PDRN promoted the proliferation of the three cell types to varying degrees, indicating that PDRN has good biocompatibility.

[0106] 2. Inhibitory effect of PDRN on JAK / STAT and NFκB inflammatory signaling pathways This invention evaluated the anti-inflammatory effect of PDRN from Example 1 using mouse peritoneal macrophages in vitro. LPS-induced inflammation models typically stimulate the levels of many inflammation-related inflammatory factors, including cytokines that may induce colorectal inflammation, such as TNF-α, IL-1β, IL-6, and Cxcl-10.

[0107] (1) Isolation of primary peritoneal macrophages 38.5 g BBL TM Thioglycolate medium (Brewer's modified powder) (BD Biosciences, catalog number: 211716) was dissolved in 1 L of purified water, stirred continuously, and autoclaved at 121°C for 15 minutes, then stored at 4°C for at least 3 months. Mice were intraperitoneally injected with 1 mL of thioglycolate. After 72 hours, the mice were necropsyed, and peritoneal macrophages were collected by flushing the peritoneal cavity.

[0108] (2) RNA extraction and RT-PCR detection Primary peritoneal macrophages were exposed to PDRN solutions from Example 1 at concentrations of 0 μg / mL, 50 μg / mL, 100 μg / mL, and 500 μg / mL for 30 minutes, followed by stimulation with 100 ng / mL LPS for 6 hours. The culture medium was discarded, and RNA was extracted from the cells. Total RNA was extracted from the cultured cells using RNAiso Plus (TaKaRa, catalog number 9109), and genomic DNA removal and reverse transcription were performed using the PrimeScript™ RT Kit (Roche, catalog number RR037A) and Genomic DNA Eraser. cDNA samples were amplified using the StepOne Plus Real-Time PCR system (Applied Biosystems) with SYBR Green PCR Master Mix. The expression levels of target genes IL-6 and TNF-α were normalized to β-actin and calculated using E=2- The relative expression level was determined by Ct relative quantification. The in vitro RT-qPCR experiment was repeated three times.

[0109] (3) Western blotting (WB) Primary peritoneal macrophages were exposed to PDRN solutions from Example 1 at concentrations of 0 mg / mL, 250 μg / mL, 500 μg / mL, and 1000 μg / mL for 2 hours, followed by stimulation with 20 ng / mL IL-6 for 10 minutes, 50 ng / mL IFN-β for 10 minutes, or 100 ng / mL LPS for 0.5 hours. Cells were then washed with cold phosphate-buffered saline (PBS) and collected in cell lysis buffer containing 1% protease and phosphatase inhibitors. The lysis supernatant, containing 20 μg of protein, was separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, catalog number 10600034). The membrane was blocked for 2 hours with 5% skim milk solution (TBST, 10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and then incubated overnight with primary antibody at 4°C. After washing three times with TBST, the mixture was incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 2 hours. The immune complexes were developed using Immobilon™ Western chemiluminescent HRP substrate (Millipore, catalog number WBKLS0500) and captured using a Tanon 5200 imaging system.

[0110] mRNA results demonstrate that the PDRN of this invention can significantly inhibit the levels of a series of inflammatory factors induced by LPS stimulation. Figure 2 (DG in the text). This invention further identifies the effect of PDRN on the activation of key regulators of LPS-induced signaling and subsequent JAK / STAT signaling induced by IL-6 and IFN-β. LPS activates the IKK kinase complex, which in turn activates IκBα / β proteins, followed by NF-κB translocation to the nucleus and initiation of transcription of downstream inflammatory genes. In patients with acute UC, STAT2 signaling becomes active, disrupting intestinal homeostasis and leading to epithelial cell necrosis. Furthermore, inhibition of STAT3 has been shown to restore TH17 / Treg cell homeostasis in the intestinal microenvironment, thereby improving the inflammatory response in acute UC. Western blot results showed ( Figure 2 PDRN significantly reduced the phosphorylation levels of IKK, STAT2, and STAT3, and inhibited the phosphorylation levels of JAK2 and JAK3, upstream signals of STAT3, suggesting that PDRN has a good inhibitory effect on both the JAK / STAT and NFκB inflammatory signaling pathways.

[0111] Example 3: PDRN improves clinical symptoms in UC model mice 1. Animal Experiment Design Animal experiment design process as follows Figure 3As shown in A in the figure. Six- to eight-week-old female C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were housed in an SPF-grade environment at a temperature of 22±1℃ and a relative humidity of 50±1%, with a 12-hour light / 12-hour dark cycle. An ulcerative colitis (UC) model was established in the mice via oral administration of DSS. All animal experiments were approved by the Laboratory Animal Committee of the College of Pharmacy, Ocean University of China.

[0112] Mice were randomly divided into five groups: normal control group (sterile water), UC model group (DSS), DJ treatment group (DSS + PN), DC treatment group (DSS + PDRN-1), and PDRN treatment group (DSS + PDRN from Example 1).

[0113] Mice in the UC model group, DJ treatment group, DC treatment group, and PDRN treatment group were orally administered 100 μL of 3% DSS in drinking water for one week (D0-D7). The normal control group was orally administered the same amount of drinking water as a control.

[0114] Normal control group: D7-D12 were administered the same amount of drinking water by gavage daily.

[0115] UC model group: D7-D12 were given the same amount of drinking water by gavage daily.

[0116] DJ treatment group: 20 mg / kg of PN (purchased from PharmaResearch Co., Ltd., catalog number DJU) solution was administered by gavage daily from D7 to D12.

[0117] DC treatment group: PDRN-1 (purchased from PharmaResearch Co., Ltd., catalog number DCELL-I) solution was administered by gavage daily from D7 to D12.

[0118] PDRN treatment group: PDRN solution of Example 1 was administered by gavage at a dose of 20 mg / kg daily from D7 to D12.

[0119] Four to eight hours after the last gavage administration on day 12, all mice were euthanized by carbon dioxide asphyxiation, and their organs, serum, and intestines were collected for qRT-PCR, RNA-Seq, and immunofluorescence analysis.

[0120] 2. PDRN improves clinical symptoms in UC model mice. 2.1 Measurement of colon length, stool condition and body weight.

[0121] Fecal status was recorded and monitored daily from days 7 to 12 for scoring the Diarrhea Index (DAI), body weight was recorded, and the percentage of body weight recovered by mice was calculated. After sacrifice, the colon was collected, macroscopic images were recorded, and colon length was quantified.

[0122] 2.1.1 DAI Scoring Criteria The DAI (Disease Activity Index) score is primarily used to assess disease activity in animal models of inflammatory bowel diseases (such as ulcerative colitis and Crohn's disease). The following are the DAI scoring criteria: Weight changes: 0 points: No weight loss or weight gain; 1 point: Weight loss of 1%-5%; 2 points: Weight loss of 5%-10%; 3 points: Weight loss of 10%-20%; 4 points: Weight loss of more than 20%.

[0123] Stool characteristics: 0 points: Normal stool (formed, dry); 1 point: Soft stool (slightly soft, formed); 2 points: Mucus-like stool (containing a small amount of mucus); 3 points: Loose stool (watery, unformed).

[0124] Fecal occult blood: 0 points: Negative (no occult blood); 1 point: Light blue / Occult blood positive (minor bleeding); 2 points: Dark blue / Occult blood obvious (moderate bleeding); 3 points: Gross blood in stool (massive bleeding).

[0125] Total score calculation: The scores of the three indicators are added together, and the average is taken as the final DAI score.

[0126] 2.1.2 Calculation of mouse weight recovery percentage The mice's weight during the recovery period was compared to their initial weight, and the percentage of recovery was calculated. The formula is as follows: Weight recovery percentage = (weight during recovery period / initial weight) × 100%; Recovery period weight: The weight (g) of the mouse during the recovery period.

[0127] Initial weight: The weight (g) of the mouse at the start of the experiment.

[0128] 2.2 Hematoxylin-eosin (H&E) staining.

[0129] Mouse colon tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Sections were dewaxed in xylene and covered with a gradient of ethanol solutions. After rinsing 3-4 times, the sections were incubated in hematoxylin solution, counterstained with eosin, dehydrated, mounted with neutral resin, dried, photographed under a fluorescence microscope, and histologically scored and the percentage of involvement determined.

[0130] Histopathological score (HS): A 1cm segment of mouse colon tissue was prepared into paraffin sections, stained with hematoxylin and eosin (HE), and the degree of inflammatory damage to the colonic mucosa was observed under a light microscope using a blind method. The score was determined according to the classic histological damage scoring criteria. 2.2.1 Histological scoring Inflammation level: Assess the extent and distribution of inflammatory cells (such as lymphocytes, neutrophils, macrophages, etc.) in the tissue. The scoring level may range from 0 (no inflammation) to 4 (severe inflammatory cell infiltration).

[0131] Degree of tissue damage or lesion: Assess the extent of damage to tissue structure, such as cell necrosis, apoptosis, fibrosis, edema, etc. The scoring level may range from 0 (no damage) to 4 (severe damage).

[0132] Total score calculation: The scores of the two indicators are added together, and the total score is taken as the final histological score.

[0133] 2.2.2 Calculation of Percentage of Affected Individuals The calculation criteria for the percentage of affected individuals are shown in Table 1: Table 1

[0134] The measurement results are as follows Figure 3 As shown: During the acute phase of UC, mice exhibited obvious clinical symptoms, including weight loss ( Figure 3 The results showed that during the acute phase of UC, the length of the colon and rectum in the UC model group was significantly shorter than that in the normal control group (D), diarrhea, and fecal occult blood. Figure 3 In the AB section, signs of edema were also observed in the colon and rectum, along with a decreased DAI score. Figure 3 All of these are manifestations of acute colorectal inflammation. Administration of the PDRN of this invention significantly reversed the shortening of the colon and rectum induced by DSS. The positive control groups, DJ and DC, showed similar effects, suggesting that the PDRN of this invention has a preliminary effect on alleviating colorectal inflammation in mice. However, the mechanism of action of the PDRN of this invention needs further verification. Histopathological examination showed abundant intestinal glands, numerous goblet cells, uniform staining of the lamina propria, and normal, regularly arranged muscle fiber morphology and structure in the normal intestinal mucosa lamina propria. In contrast, during the acute phase of UC, the UC model group showed mucosal erosion, shortened crypts, edema, and a large number of inflammatory cell infiltrations in the mucosa and lamina propria. Administration of the PDRN of this invention can alleviate the histological damage caused by DSS, manifested as increased goblet cell density and increased intestinal mucosal cell density. The cell density was higher, the intestinal mucosa was intact, and the histological score and inflammatory cell infiltration were reduced. Figure 3 Similar effects were also observed in the EG group (positive drug DJ group and DC group), and these observations provide theoretical support for the PDRN resistance to acute UC of the present invention.

[0135] 3. Effects of PDRN on the expression of inflammation-related proteins in UC mice 3.1 Immunohistochemical staining Sections were placed in a 4°C humidified chamber and incubated overnight with primary antibody prepared at a 1:500 ratio (anti-CD11b or IL-1β). Sections were washed three times with PBS (pH 7.4) with shaking for 5 minutes each time, followed by incubation with fluorescent CY3 secondary antibody (Servicebio, GB21303) at room temperature in the dark for 50 minutes. Sections were then incubated with DAPI solution (Servicebio, G1012) at room temperature in the dark for 10 minutes, followed by incubation with an autofluorescence burst reagent (Servicebio, G1221) for 5 minutes. Finally, sections were blocked with an anti-fading blocking agent (Servicebio, G1401). Microscopic examination and image acquisition were performed using a fluorescence microscope (Nikon, NIKON ECLIPSE C1). Fluorescence color channels were adjusted using CaseViewer software. Positive results were calculated using Servicebio®'s Aipathwell® digital pathology image analysis software.

[0136] 3.2 Flow cytometry determination of CD45+ and MCHII / 7AAD expression levels 3.2.1 Sample Preparation Cells were collected from the lamina propria of mouse colorectal tissue, homogenized, diluted with an equal volume of PBS, filtered through a 100 μm filter, washed with 5 volumes of flow cytometry buffer, centrifuged at 300×g for 5 minutes, and the supernatant was discarded. The washing process was repeated twice. Cells were resuspended in flow cytometry buffer, counted, and the concentration adjusted to 1×10⁻⁶. 6 -5×10 6 Cells / mL.

[0137] 3.2.2 Antibody staining (surface markers CD45 and MHCII) Grouping and sample loading: Take flow cytometry tubes and label them "Blank Control", "Isotype Control", and "CD45". + The groups were categorized into "single staining", "MHCII single staining", and "CD45 / MHCII / 7AAD triple staining". 100 μL of single-cell suspension (approximately 1 × 10⁻⁶ cells) was added to each group. 6 -5×10 6 (Cells). Antibody incubation: Add CD45-fluorescent antibody and MHCII-fluorescent antibody (CD45-PE 5 μL + MHCII-APC 3 μL) to the concentrations recommended in the antibody instructions; add the corresponding isotype control antibody (such as IgG1-PE + IgG2a-APC) to the isotype control tubes, and add only buffer to the blank control tubes; mix gently and incubate at 4°C in the dark for 20-30 minutes.

[0138] Washing: Add 2 mL of flow cytometry buffer to each tube, centrifuge at 300×g for 5 minutes, discard the supernatant, and repeat the washing once to avoid antibody residue.

[0139] 3.2.3 7AAD staining 7AAD incubation: Resuspend the cell pellet in 200 μL of flow cytometry buffer, add 7AAD dye to a final concentration of 0.5 μg / mL; incubate at room temperature in the dark for 5-15 minutes, then analyze using the flow cytometry.

[0140] 3.2.4 Flow cytometry detection (1) Instrument preparation: Turn on the flow cytometer, preheat and calibrate (use fluorescent microspheres to calibrate the optical path and voltage), and set the detection channels: CD45-PE (Detection channel PE: 561 nm excitation, 585 / 42 nm emission); MHCII-APC (Detection Channel APC: 633 nm excitation, 660 / 20 nm emission); 7AAD (Detection channel APC-Cy7 or PerCP-Cy5.5: 633 nm excitation, 780 / 60 nm emission, the specific frequency should be adjusted according to the instrument configuration).

[0141] (2) Sample loading and data acquisition: Place the stained sample tubes on the sample stage and collect 1×10⁻⁶ samples from each tube. 4 -1×10 5 Individual cells (adjusted according to cell concentration); Record the scattered light signal (FSC / SSC) and fluorescence signal (CD45, MHCII, 7AAD) for each group.

[0142] 3.2.5 Data Analysis (1) Gate strategy: Step 1: Set gates for FSC-A vs SSC-A to exclude debris and adherents (adhesives can be further excluded by FSC-H vs FSC-W); Step 2: 7AAD - Live cell phylogenetics: In the scatter plot of "SSC-A vs 7AAD", circle 7AAD.- Cells (living cells); Step 3: CD45 + Cell phylogenetics: In living cells, CD45 is phylogenetically defined by "CD45 fluorescence intensity vs SSC-A". + Cell population; Step 4: MHCII Expression Analysis: On CD45 + / 7AAD - In a live cell population, the positive rate of MHCII (%MHCII) was analyzed. + ) and mean fluorescence intensity (MFI).

[0143] (2) Calculation of results: CD45 + Cell ratio = (CD45) + (Number of cells / Total number of viable cells) × 100%; MHCII expression level = CD45 + / 7AAD - MHCII in cells + Positive rate and MFI; 7AAD + dead cell ratio = (7AAD) + Cell count (total cell count) × 100% (used to assess sample viability).

[0144] Activation of the NF-κB signaling pathway is the core mechanism of the pro-inflammatory effect of IL-1β. In UC patients, IL-1β binds to the IL-1 receptor, releasing nuclear factor κB (NF-κB) into the cell nucleus. Activated NF-κB upregulates the expression of various pro-inflammatory factors (such as TNF-α, IL-6, and IL-8) and chemokines, thereby enhancing the inflammatory response. cD11b (also known as integrin αM or Mac-1) is mainly expressed on the surface of myeloid cells (such as neutrophils, monocytes, and macrophages) and plays an important role in regulating immune imbalance, mucosal barrier disruption, and persistent inflammation in UC. The results showed that, compared with the normal control group, the expression of IL-1β and CD11b proteins was significantly increased in the UC model group. Figure 4 A in the text). Immunofluorescence analysis ( Figure 4 The BC assay showed that the PDRN of this invention significantly reduced the increase of these inflammatory proteins induced by DSS, suggesting that PDRN prevents UC-induced intestinal inflammatory storm by inhibiting inflammatory proteins. Furthermore, the anti-inflammatory effect of the PDRN of this invention was stronger than that of the DJ and DC groups. In addition, flow cytometry (BC) Figure 4Analysis of intestinal lamina propria cells using DE (diethyltoluamide) assays showed that the number of CD45+ immune cells in the intestinal mucosa of mice in the colitis model group was significantly increased compared to the control group (p<0.05), while the number of CD45+ immune cells in the PDRN group of this invention was significantly reduced compared to the UC model group. Furthermore, myeloid cells (including macrophages and dendritic cells) in the intestinal mucosa also showed a significant activation state; for example, MCHII / 7AAD was statistically downregulated in the PDRN treatment group compared to the UC model group (p<0.05). These results suggest that in colitis, the intestinal immune environment exhibits enhanced Th17 cell-mediated immune responses and an imbalance in immune regulatory mechanisms. These data not only reveal the immunopathological characteristics of colitis but also provide an experimental basis for further exploration of therapeutic strategies targeting Th17 cells and regulatory T cells.

[0145] 4. Transcriptome sequencing analysis showed that PDRN exhibited excellent anti-inflammatory effects in the UC model. In this experiment, colorectal tissue samples were collected from untreated (normal control group), colitis model only (UC model group), DJ treatment group, DC treatment group, and PDRN treatment group mice. Total RNA was extracted from cells in each group using RNAiso Plus. RNA quality was measured using a 5300 Bioanalyzer (Agilent), and quantification was performed using an ND-2000 (NanoDrop Technologies). Sequencing libraries were constructed using the NovaSeq X Plus sequencing platform (PE150). Clean reads from each sample were aligned to a specified reference genome sequence (Mus_musculus, GRCm39). Based on the quantitative expression data, differentially expressed genes were analyzed between groups to screen for genes with significant expression differences. The differential analysis software was DESeq2, and the screening threshold was |log2FC|>= 2 and padjust<0.05. KEGG / GO pathway enrichment analysis was performed on the differentially expressed genes. gSEzzz has been uploaded to GEO.

[0146] This invention performed transcriptomic analysis to explore the therapeutic potential of the PDRN of this invention for colitis. The results showed ( Figure 5 In DSS-treated colon tissue, the expression of numerous genes was altered, and KEGG functional enrichment analysis identified various upregulated pro-inflammatory signaling pathways, such as the TNF signaling pathway and the Il-17 signaling pathway. Similar results were observed when RNA was extracted from colon tissue and the relative expression of mRNA was detected. Figure 5(BE in the text). Of particular note is that activation of the TNF signaling pathway and the IL-17 signaling pathway (both known anti-inflammatory pathways) was more pronounced in the PDRN compound treatment group samples of this invention. In summary, this study demonstrates that the PDRN compound of this invention significantly inhibits inflammation in a colitis model by regulating key signaling pathways and inflammatory response genes. These findings provide a scientific basis for the PDRN compound of this invention as a potential therapeutic agent for colitis.

[0147] 5. Effects of PDRN on gut microbiota in UC model mice DNA Extraction and 16S rDNA Sequencing Analysis: Following the instructions of the FastPure Fecal DNA Extraction Kit (MJYH, Shanghai, China), total genomic DNA of the microbial community was extracted from fecal samples. The integrity of the extracted genomic DNA was assessed by 1% agarose gel electrophoresis, and the concentration and purity of the DNA were determined using a NanoDrop2000 (Thermo Scientific, USA). DNA samples were stored at -20°C for subsequent experiments. Library construction was performed using the SMRTbell prep kit 3.0. Sequencing was performed using the PacBio Sequel IIe system (Shanghai Meiji Biomedical Technology Co., Ltd.). HiFi sequences were generated from the sequenced subsequences using the CCS mode of SMRT-Linkv11.0 for subsequent data analysis.

[0148] The pathogenesis of colorectal diseases is closely related to the host's gut microbiota dysbiosis. To comprehensively study the effects of DSS and the PDRN treatment described in Example 1 of this invention on the gut microbiota, this invention used 16S rRNA sequencing to analyze the microbial composition of the colonic contents. This invention found ( Figure 6 In group B), the normal control group had the highest number of operational taxa (ASVs). DSS modeling disrupts the ASV numbers of this microorganism, while drug administration reverses this trend, making the PDRN group closer to the normal control group (NC group). This invention used principal coordinate analysis (PCoA) to perform β-diversity analysis at the species level. Figure 6 (A) The results showed that the control group was tightly clustered and significantly separated from the DSS group, while the PDRN group of this invention was located between the control and DSS groups. α-diversity was assessed by ACE and Chao1 indices, which reflect the richness and diversity of the community. It can be seen that after DSS administration, ACE and Chao1 indices decreased significantly, indicating that DSS stimulation significantly affected the richness and diversity of the mouse gut microbiota community (A). Figure 6(DE in the text). Unexpectedly, PDRN intake in this invention significantly improved these indices, suggesting that the PDRN in this invention may play a role in improving DSS-induced dysbiosis of microbial species. The MDI index is a commonly used indicator to measure the degree of microbial ecological dysbiosis; the higher the value, the more severe the dysbiosis. Figure 6 The results in C show that DSS led to an increase in MDI, while the PDRN administration of this invention downregulated this index. Among them, *Parvibacter* typically produces succinic acid in vivo, maintaining intestinal metabolism and function; *Lachnospiraceae_NK4A136_group* is a beneficial bacterium that can ferment to produce small molecule acids; and *Adlercreutzia* typically has anti-inflammatory effects. In the genus-level analysis, DSS led to a significant reduction in the number of the above-mentioned bacterial colonies, but their abundance recovered somewhat after treatment with the PDRN of this invention. Figure 7 In summary, the results suggest that the PDRN of this invention may have a beneficial effect on ulcerative colitis caused by DSS by regulating the abundance and diversity of gut microbiota and increasing the abundance of beneficial bacteria.

[0149] Comparative Example 1: Preparation of PDRN PDRN was prepared using an enzymatic hydrolysis method, and the specific steps are as follows: S1. After washing the thawed salmon sperm with physiological saline, use a high-speed blender to add physiological saline at a solid-liquid ratio of 1:6 to fully break down the salmon sperm and obtain a solid-liquid mixture. S2. Centrifuge the solid-liquid mixture at 4℃ and 3500rpm for 30min, discard the supernatant, add 100g of precipitate to 500mL of mixed salt solution (0.15M NaCl / 0.5 mM EDTA) at 37℃, and react with stirring for 10min. S3. Add the compound enzyme at a ratio of 250:1 (mass of salmon sperm to mass of trypsin), and hydrolyze at 37°C with stirring for 20 h to obtain the hydrolysate. S4. After enzymatic hydrolysis, add 10% SDS by weight of salmon sperm and NaCl to the hydrolysate, and continue stirring for 4 h. Collect the supernatant, centrifuge at 6000 rpm for 1 h at room temperature, and collect the supernatant.

[0150] S5. Add pre-cooled anhydrous ethanol to the supernatant at a volume ratio of 2:1, stir, and precipitate flocculated PDRN at 4℃. Wash twice with 75% ethanol, centrifuge at 4000 rpm for 5 min, discard the supernatant, remove the residual ethanol with a pipette tip, crush the precipitate, and freeze-dry it in a freeze dryer to obtain PDRN. After freeze-drying, dissolve it in physiological saline to obtain PDRN solution.

[0151] Comparative Example 2: Preparation of PDRN PDRN was prepared using an enzymatic hydrolysis method, and the specific steps are as follows: S1. After washing the thawed salmon sperm with physiological saline, use a high-speed blender to add physiological saline at a solid-liquid ratio of 1:6 to fully break down the salmon sperm and obtain a solid-liquid mixture. S2. Centrifuge the solid-liquid mixture at 4℃ and 3500rpm for 30min, discard the supernatant, add 100g of precipitate to 500mL of mixed salt solution (0.15M NaCl / 0.5 mM EDTA) at 37℃, and react with stirring for 10min. S3. Add the compound enzyme at a ratio of 250:1 (mass of salmon sperm to papain) and hydrolyze at 37°C with stirring for 20 h to obtain the hydrolysate. S4. After enzymatic hydrolysis, add 10% SDS by weight of salmon sperm and NaCl to the hydrolysate, and continue stirring for 4 h. Collect the supernatant, centrifuge at 6000 rpm for 1 h at room temperature, and collect the supernatant.

[0152] S5. Add pre-cooled anhydrous ethanol to the supernatant at a volume ratio of 2:1, stir, and precipitate flocculated PDRN at 4℃. Wash twice with 75% ethanol, centrifuge at 4000 rpm for 5 min, discard the supernatant, remove the residual ethanol with a pipette tip, crush the precipitate, and freeze-dry it in a freeze dryer to obtain PDRN. After freeze-drying, dissolve it in physiological saline to obtain PDRN solution.

[0153] Preparation of PDRN (Comparative Example 3) PDRN was prepared using an enzymatic hydrolysis method, and the specific steps are as follows: S1. After washing the thawed salmon sperm with physiological saline, use a high-speed blender to add physiological saline at a solid-liquid ratio of 1:6 to fully break down the salmon sperm and obtain a solid-liquid mixture. S2. Centrifuge the solid-liquid mixture at 4℃ and 3500rpm for 30min, discard the supernatant, add 100g of precipitate to 500mL of mixed salt solution (0.15M NaCl / 0.5 mM EDTA) at 37℃, and react with stirring for 10min. S3. Add the compound enzyme at a ratio of 250:1 (mass ratio of salmon sperm to compound enzyme (proteinase K:papain of 2.5:1) and hydrolyze at 37°C for 20 h with stirring to obtain the enzymatic hydrolysate. S4. After enzymatic hydrolysis, add 10% SDS by weight of salmon sperm and NaCl to the hydrolysate, and continue stirring for 4 h. Collect the supernatant, centrifuge at 6000 rpm for 1 h at room temperature, and collect the supernatant.

[0154] S5. Add pre-cooled anhydrous ethanol to the supernatant at a volume ratio of 2:1, stir, and precipitate flocculated PDRN at 4℃. Wash twice with 75% ethanol, centrifuge at 4000 rpm for 5 min, discard the supernatant, remove the residual ethanol with a pipette tip, crush the precipitate, and freeze-dry it in a freeze dryer to obtain PDRN. After freeze-drying, dissolve it in physiological saline to obtain PDRN solution.

[0155] Comparative Example 4: Preparation of PDRN PDRN was prepared using an enzymatic hydrolysis method, and the specific steps are as follows: S1. After washing the thawed salmon sperm with physiological saline, use a high-speed blender to add physiological saline at a solid-liquid ratio of 1:6 to fully break down the salmon sperm and obtain a solid-liquid mixture. S2. Centrifuge the solid-liquid mixture at 4℃ and 3500rpm for 30min, discard the supernatant, add 100g of precipitate to 500mL of mixed salt solution (0.15M NaCl / 0.6 mM EDTA) at 37℃, and react with stirring for 10min. S3. Add the compound enzyme at a ratio of 250:1 (mass ratio of salmon sperm to compound enzyme (proteinase K:papain in a mass ratio of 2.5:1) and hydrolyze at 37°C for 20 h with stirring to obtain the enzymatic hydrolysate. S4. After enzymatic hydrolysis, add 10% SDS by weight of salmon sperm and NaCl to the hydrolysate, and continue stirring for 4 h. Collect the supernatant, centrifuge at 6000 rpm for 1 h at room temperature, and collect the supernatant.

[0156] S5. Add pre-cooled anhydrous ethanol to the supernatant at a volume ratio of 2:1, stir, and precipitate flocculated PDRN at 4℃. Wash twice with 75% ethanol, centrifuge at 4000 rpm for 5 min, discard the supernatant, remove the residual ethanol with a pipette tip, crush the precipitate, and freeze-dry it in a freeze dryer to obtain PDRN. After freeze-drying, dissolve it in physiological saline to obtain PDRN solution.

[0157] Preparation of PDRN (Comparative Example 5) PDRN was prepared using an enzymatic hydrolysis method, and the specific steps are as follows: S1. After washing the thawed salmon sperm with physiological saline, use a high-speed blender to add physiological saline at a solid-liquid ratio of 1:6 to fully break down the salmon sperm and obtain a solid-liquid mixture. S2. Centrifuge the solid-liquid mixture at 4℃ and 3500rpm for 30min, discard the supernatant, add 100g of precipitate to 500mL of mixed salt solution (0.15M NaCl / 0.6 mM EDTA) at 37℃, and react with stirring for 10min. S3. Add the compound enzyme at a ratio of 250:1 (mass ratio of salmon sperm to compound enzyme (trypsin:papain in a mass ratio of 2.5:1) and hydrolyze at 37°C for 20 h with stirring to obtain the enzymatic hydrolysate. S4. After enzymatic hydrolysis, add 10% SDS by weight of salmon sperm and NaCl to the hydrolysate, and continue stirring for 4 h. Collect the supernatant, centrifuge at 6000 rpm for 1 h at room temperature, and collect the supernatant.

[0158] S5. Add pre-cooled anhydrous ethanol to the supernatant at a volume ratio of 2:1, stir, and precipitate flocculated PDRN at 4℃. Wash twice with 75% ethanol, centrifuge at 4000 rpm for 5 min, discard the supernatant, remove the residual ethanol with a pipette tip, crush the precipitate, and freeze-dry it in a freeze dryer to obtain PDRN. After freeze-drying, dissolve it in physiological saline to obtain PDRN solution.

[0159] Preparation of PDRN (Comparative Example 6) PDRN was prepared using an enzymatic hydrolysis method, and the specific steps are as follows: S1. After washing the thawed salmon sperm with physiological saline, use a high-speed blender to add physiological saline at a solid-liquid ratio of 1:6 to fully break down the salmon sperm and obtain a solid-liquid mixture. S2. Centrifuge the solid-liquid mixture at 4℃ and 3500rpm for 30min, discard the supernatant, add 100g of precipitate to 500mL of mixed salt solution (0.15M NaCl / 0.5 mM EDTA) at 37℃, and react with stirring for 10min. S3. Add the compound enzyme at a ratio of 250:1 (mass of salmon sperm to mass of compound enzyme (mass ratio of trypsin:papain of 1:1) and hydrolyze at 37°C for 20 h with stirring to obtain the enzymatic hydrolysate. S4. After enzymatic hydrolysis, add 10% SDS by weight of salmon sperm and NaCl to the hydrolysate, and continue stirring for 4 h. Collect the supernatant, centrifuge at 6000 rpm for 1 h at room temperature, and collect the supernatant.

[0160] S5. Add pre-cooled anhydrous ethanol to the supernatant at a volume ratio of 2:1, stir, and precipitate flocculated PDRN at 4℃. Wash twice with 75% ethanol, centrifuge at 4000 rpm for 5 min, discard the supernatant, remove the residual ethanol with a pipette tip, crush the precipitate, and freeze-dry it in a freeze dryer to obtain PDRN. After freeze-drying, dissolve it in physiological saline to obtain PDRN solution.

[0161] Experimental Example 1: Inhibitory effect of PDRN on JAK / STAT and NFκB inflammatory signaling pathways in Comparative Examples 1-6 Primary peritoneal macrophages were exposed to PDRN solutions at concentrations of 500 mg / mL (Comparative Examples 1-6) for 30 minutes, followed by stimulation with 100 ng / mL LPS for 6 hours. The culture medium was discarded, and RNA was extracted from the cells. Total RNA was extracted from the cultured cells using RNAiso Plus (TaKaRa, catalog number 9109), and genomic DNA removal and reverse transcription were performed using the PrimeScript™ RT Kit (Roche, catalog number RR037A) and Genomic DNA Eraser. cDNA samples were amplified using the StepOne Plus Real-Time PCR system (Applied Biosystems) with SYBR Green PCRMaster Mix. The expression level of the target gene IL-6 was normalized to β-actin and calculated using E=2- The relative expression level was determined by Ct relative quantification. The in vitro RT-qPCR experiment was repeated three times.

[0162] The measurement results are as follows Figure 8 As shown, although the PDRN of Comparative Examples 1-6 can reduce the IL-6 expression level to a certain extent, the effect is not as obvious as the PDRN of Example 1 of the present invention in reducing IL-6 expression.

[0163] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A PDRN, characterized in that, The preparation method of the PDRN comprises the following steps: S1, after adding a solvent to break the salmon sperm, a solid-liquid mixture is obtained; S2, the solid-liquid mixture is centrifuged, the precipitate is collected, a mixed salt solution is added, and a reaction liquid is obtained; S3, a complex enzyme is added to the reaction liquid, and enzymolysis is carried out to obtain an enzymolysis liquid; S4, the enzymolysis liquid is removed after the residual impurities are removed, and after alcohol precipitation, the precipitate is collected, washed, centrifuged, and collected, the precipitate is broken, and freeze-drying is carried out to obtain PDRN; The mixed salt solution in step S2 is a mixed solution of NaCl and EDTA, and the molar ratio of NaCl to EDTA in the mixed salt solution is 0.1-0.5:1; The complex enzyme in step S3 is a mixture of trypsin and papain, and the mass ratio of trypsin to papain is 2-3:

1.

2. The PDRN of claim 1, wherein, The solvent in step S1 includes any one or more of physiological saline, water, phosphate buffer, citrate buffer, acetate buffer, HEPES buffer, Tris-HCl buffer, and Tris buffer.

3. The PDRN of claim 1, wherein, The amount of the solvent added in step S1 is 5-10 mL of the solvent per 1 g of salmon sperm.

4. The PDRN of claim 1, wherein, The molar ratio of NaCl to EDTA in the mixed salt solution in step S2 is 0.3:

1.

5. The PDRN of claim 1, wherein, The amount of the mixed salt solution added in step S2 is 3-7 mL of the mixed salt solution per 1 g of the precipitate in step S2.

6. The PDRN of claim 1, wherein, The reaction time in step S2 is 5-15 min.

7. The PDRN of claim 1, wherein, The mass ratio of trypsin to papain in step S3 is 2.5:

1.

8. The PDRN of claim 1, wherein, The mass ratio of the complex enzyme in step S3 to the salmon sperm in step S1 is 1:200-300.

9. The PDRN of claim 1, wherein, The reaction conditions of the enzymolysis in step S3 are 35-40°C for 15-25 h.

10. The PDRN of claim 1, wherein, The step of removing the residual impurities after the enzymolysis in step S4 comprises adding SDS and NaCl to the enzymolysis liquid, and then centrifuging to collect the supernatant; and the alcohol precipitation step in step S4 comprises adding ethanol to the supernatant.

11. The PDRN of claim 10, wherein, The amount of SDS added is 10%-15% of the mass of the salmon sperm; and the final concentration of NaCl is 1.5-2.0 M.

12. The PDRN of claim 10, wherein, The ethanol is 90%-100% v / v ethanol; and the volume ratio of the ethanol to the supernatant is 1-3:

1.

13. The preparation method of the PDRN according to any one of claims 1-12.

14. Use of PDRN according to any one of claims 1-12 for the preparation of a product, characterized in that, The product is a drug for preventing, treating, or assisting in treating colitis.

15. Use according to claim 14, characterized in that, The colitis includes any one or more of infectious colitis, ischemic colitis, pseudomembranous colitis, ulcerative colitis, and Crohn's disease of the colon.

16. A medicine, characterized in that, The drug comprises the PDRN according to any one of claims 1-12.

17. The pharmaceutical product according to claim 16, characterized in that The drug further comprises a pharmaceutically acceptable excipient.

18. The pharmaceutical product according to claim 17, characterized in that The dosage form of the drug comprises a gastrointestinal administration dosage form or a non-gastrointestinal administration dosage form.

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